Ouabain Suppresses Glucose-Induced Mitochondrial ATP Production and Insulin Release by Generating Reactive Oxygen Species in Pancreatic Islets

Abstract

We examined the effects of reduced Na ؉ /K ؉ -ATPase activity on mitochondrial ATP production and insulin release from rat islets. Ouabain, an inhibitor of Na ؉ /K ؉ -ATPase, augmented 16.7 mmol/l glucose-induced insulin release in the early period but suppressed it after a delay of 20 -30 min. Unexpectedly, the ATP content in an islet decreases in the presence of 16.7 mmol/l glucose when Na ؉ /K ؉ -ATPase activity is diminished by ouabain, despite the reduced consumption of ATP by the enzyme. Ouabain also suppressed the increment of ATP content produced by glucose even in Ca 2؉ -depleted or Na ؉ -depleted conditions. That mitochondrial membrane hyperpolarization and O 2 consumption in islets exposed to 16.7 mmol/l glucose were suppressed by ouabain indicates that the glycoside inhibits mitochondrial respiration but does not produce uncoupling. Ouabain induced mitochondrial reactive oxygen species (ROS) production that was blocked by myxothiazol, an inhibitor of site III of the mitochondrial respiratory chain. An antioxidant, ␣-tocopherol, also blocked ouabain-induced ROS production as well as the suppressive effect of ouabain on ATP production and insulin release. However, ouabain did not directly affect the mitochondrial ATP production originating from succinate and ADP. These results indicate that ouabain suppresses mitochondrial ATP production by generating ROS via transduction, independently of the intracellular cationic alternation that may account in part for the suppressive effect on insulin secretion. efficacy in stimulation-secretion coupling, which also is dependent on the accelerated glucose metabolism that correlates with increments in the ATP/ADP ratio. Regulation of the ATP level in pancreatic ␤-cells, therefore, plays a crucial role in insulin secretion. Na ϩ /K ϩ -ATPase is involved in maintaining Na ϩ and K ϩ gradients across the ␤-cell plasma membrane and is thought to consume a large amount of ATP in the maintenance of homeostasis (2,3). Accordingly, the role of Na ϩ / K ϩ -ATPase in the regulation of the intracellular ATP levels in ␤-cells is of interest. We have reported that the ATP content of an islet unexpectedly decreases in the presence of glucose when Na ϩ /K ϩ -ATPase activity is diminished by ouabain, despite reduced consumption of ATP by the enzyme, whereas decrease of ATP content is not observed using thapsigargin, an inhibitor of another ATP consumer, the Ca 2ϩ -ATPase in endoplasmic reticulum (4,5). Inhibition by ouabain of glucose oxidation (6) and glucose utilization (7) has been reported previously in rat islets. However, the effect of ouabain on glucose-induced insulin release is complex. Ouabain has a stimulatory effect on glucose-induced insulin secretion in the early phase (8 -10), probably owing to increased Ca 2ϩ influx (10 -12). Such increased Ca 2ϩ influx by ouabain should be due to depolarization, since the electrogenic effect of Na ϩ /K ϩ -ATPase is to hyperpolarize the membrane potential (2). On the other hand, ouabain decreases glucoseinduced insulin release in th...

Src activation generates reactive oxygen species and impairs metabolism–secretion coupling in diabetic Goto–Kakizaki and ouabain-treated rat pancreatic islets

“…In a previous study, we found that ouabain reduces not only the increment in ATP content and the hyperpolarisation of mitochondrial membrane potential by glucose, but also the increment in O 2 consumption by glucose [17]. Since increased O 2 consumption occurs in uncoupling [38], ouabain-induced suppression of mitochondrial ATP production clearly is not mediated by uncoupling, and the suppression may derive from direct or indirect effects on the respiratory chain.…”

“…We have found that ouabain decreases glucose-induced insulin release by reducing ATP content [17]. In addition, high glucose-induced hyperpolarisation of mitochondrial membrane potential was inhibited by ouabain.…”

“…Fluorescence measurement of ROS production and change in mitochondrial membrane potential ROS production and change in mitochondrial membrane potential (ΔΨ m ) in dispersed islet cells under Ca 2+ -free conditions were measured by 5-(and 6)-chloromethyl-2′,7′-dichlorofluorescein (CM-DCF) fluorescence and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1) fluorescence, respectively, as previously reported [17]. Fluorescence was corrected by subtracting parallel blanks in which islet cells were not loaded with probes, and is presented as a ratio with respect to the value at time zero.…”

“…Measurement of insulin release, insulin content and DNA content Insulin release from cultured islets was monitored using static incubation as described previously [17]. After an aliquot of incubation medium for insulin assay was taken, the islets remaining were lysed to determine insulin and DNA contents as described previously [22].…”

Islet transplantation and antioxidant management: A comprehensive review

“…An o ther impo r ta nt po in t which ca n lead to worsening of this condition is that β cells contain low levels of antioxidative enzymes such as CAT, SOD and GPx and thus they can only weakly defend against oxidative stress [10][11][12] . These findings have demonstrated the major destructive role of oxidative stress in islet transplantation and have encouraged investigators to use antioxidants during the isolation phase and the entire transplantation process to overcome the final lack of healthy islets.…”